Polyhydroxyalkanoates (PHAs) are a broad class of polyesters that are formed naturally in many species of bacteria as storage materials for carbon, energy and reducing equivalents. These biological compounds have drawn significant scientific and industrial interest because of their potential to be utilized as biodegradable plastics. The properties of the plastic that may be produced vary depending on the lengths and types of monomer units present in the polymer, and whether these monomer units are identical, forming a homopolymer, or different, forming a random or block copolymer. Almost all PHAs studied to date may be categorized as either PHA.sub.MCL (polymers formed from medium chain length, 6-14 carbon precursors) or PHA.sub.SCL (polymers formed from short chain length, 3-5 carbon precursors) (S. Y. Lee, Biotechnol. Bioeng., 49, 1-14 (1996); see FIG. 1). Polyhydroxybutyrate (PHB) and polyhydroxyvalerate (PHV) are examples of PHA.sub.SCL, whereas polyhydroxyhexanoate (PHH) and polyhydroxyoctanoate (PHO) are examples of PHA.sub.MCL. PHA.sub.MCL polymers and copolymers also have properties superior to pure PHB (K. D. Gagnon et al, Macromolecules, 25, 3723-3728 (1992)).
The type of polyhydroxyalkanoate (PHA) polymerase (also commonly referred to as PHA synthase or, occasionally, PHA synthetase) that a microorganism possesses determines whether it can synthesize PHA.sub.SCL or PHA.sub.MCL, but there is little crossover between the two classes. A. Steinbuchel, "Polyhydroxyalkanoic Acids" in: Byrom, D. et al., Eds., Biomaterials: Novel Materials from Biological Sources, Stockton Press, New York, 263-284 (1991). Bacterial PHA polymerases accordingly fall into two categories: those that catalyze polymer formation from short chain length (C.sub.3 -C.sub.5) monomers, known as "Class I" PHA polymerases or, alternatively, PHA.sub.SCL polymerases, and those that catalyze polymer formation from long chain length (C.sub.6 -C.sub.14) monomers, known as "Class II" PHA polymerases or, alternatively, PHA.sub.MCL polymerases.
Ralstonia eutropha (until recently known as Alcaligenes eutrophus) and Pseudomonas oleovorans are two well-studied microorganisms that possess PHA polymerases that fall into this typical pattern. The biosynthetic pathway from R. eutropha is essentially limited to synthesizing polyhydroxybutyrate (PHB), a PHA.sub.SCL made from C4 monomers. The enzymes needed for PHB synthesis in R. eutropha include .beta.-ketothiolase, acetoacetyl-CoA reductase, and a PHA.sub.SCL polymerase commonly referred to as a polyhydroxybutyrate (PHB) synthase. These enzymes are encoded by the genes phbA, phbB and phbC, respectively, and are clustered into a single phbCAB operon (FIG. 2). A .sigma..sup.70 -like promoter sequence precedes the transcription start site, which is 307 bp upstream of phbC. The PHB polymerase from R. eutropha links 3-hydroxybutyryl moieties (100% relative activity) or 3-hydroxyvaleryl moieties (7.5% relative activity) to existing polyester molecules using ester bonds. It is stereospecific for D(-) stereoisomers and does not react with L(+) stereoisomers (G. W. Haywood et al., FEMS Microbiol. Lett., 57, 1-6 (1989)).
The P. oleovorans operon contains two polymerases genes bracketing gene encoding a depolymerase (FIG. 2). The two polymerase genes, phaC1 and phaC2, exhibit 37.8% and 39.5% identity with phbC from R. eutropha, respectively. Both phaC1 and phaC2 are able to complement P. putida PHA-negative mutant GPp104 to make PHA (G. Huisman et al., J. Biol. Chem., 266, 2191-2198 (1991). Both PHA polymerases isolated from P. oleovorans are PHA.sub.MCL polymerases that form polymers from medium chain length monomers. In general, the 3-hydroxylated form of whatever medium chain monomer which is supplied as carbon source is incorporated into PHA.sub.MCL. This includes monomers containing unsaturated, branched, aromatic, and halogenated groups. For example, PHA with as many as six different types of monomer units having 6-11 carbons were produced in P. oleovorans grown on C6-C10 n-alkanoic acids (H. Brandl et al., Appl. Environ. Microbiol., 54, 1977-1982 (1988)). When P. oleovorans is grown on C6 to C12 n-alkanes and 1-alkenes, PHA.sub.MCL containing saturated and saturated plus unsaturated monomers is synthesized (R. G. Lagaveen et al., Appl. Environ. Microbiol., 54, 2924-2932 (1988)).
The PHB polymerase of R. eutropha has been expressed in recombinant P. oleovorans in an attempt to produce a copolymer incorporating both short chain length and medium chain length monomers (A. Timm et al., Appl. Environ. Microbiol., 56, 3360-3367 (1990)). The transformed strain was grown on octanoate and did accumulate a polymer comprising 3HB (3-hydroxybutyrate) and 3HO (3-hydroxyoctanoate) as main constituents and 3HH (3-hydroxyhexanoate) as a minor constituent. However, the polymer was not a true copolymer or terpolymer of a short chain length (3HB) and one or more medium chain length (3HO or 3HH) monomers; rather, it was shown to be a blend (i.e., a physical mixture) of a PHB homopolymer (i.e., a PHA.sub.SCL homopolymer) and a poly(3HO-co-3HH) copolymer (i.e., a PHA.sub.MCL copolymer).
Only a few exceptions to the observed division of substrate specificities in PHA polymerases are known. Small amounts of 3-hydroxybutyrate (a C4 monomer) have been detected in PHA consisting mostly of PHA.sub.MCL produced by P. resinovorans (B. A. Ramsay et al., Appl. Environ. Biotech. 58, 744-746 (1992)). PHA.sub.MCL polymerase from P. aeruginosa and other pseudomonads of rRNA homology group I have been reported to incorporate 3-hydroxyvalerate (3HV, a C5 monomer) into PHA, but only if the cells are cultivated with valeric acid as sole carbon source. A. Timm et al., Appl. Environ. Microbiol., 56, 3360-3367 (1990). Indeed, all of the fluorescent pseudomonads belonging to rRNA homology group I are able to synthesize PHA.sub.MCL whose composition is dependent upon the length of the carbon backbone in the substrate supplied (S. Y. Lee et al., Can. J. Microbiol., 41, (suppl. 1), 207-215 (1995)). Pseudomonas sp. 61-3 is reported to produce a PHB homopolymer and a P(3HB-co-3HA) copolymer when glucose is presented as a substrate under nitrogen-free conditions. The copolymer consisted of a mixture of 44 mol % C4 monomer, with the rest of the polymer distributed among C6-C12 monomers (M. Kato et al., Appl. Microbiol. Biotech., 44, 1-8 (1996)). This strain seems to have two polymerases, and the one with seemingly broader specificity may operate only under nitrogen-free conditions. P. putida strain GP4BH1 had been shown to accumulate polyester containing 3HB and 3HA.sub.MCL when grown on various carbon sources (Steinbuchel et al., Appl. Microbiol. Biotech., 37, 691-697 (1992)). This was the first naturally occurring bacterium to be described that accumulates polyester containing 3HB and 3HO at significant levels. However, this was not a true copolymer, but instead a blend (i.e., a mixture of two homopolymers) that was probably formed because of the existence of more than one polymerase in this organism.
PHA polymerases able to reliably catalyze incorporation of a broader range of precursor lengths would be extremely useful since novel PHA copolymers with unique composition and properties could be synthesized. Actual attempts to broaden the substrate specificities of bacterial PHA polymerases have thus far been confined to manipulations of the carbon source supplied to the polymer-synthesizing microorganism. Fusion or chemical mutagenesis of R. eutropha PHB polymerase and P. oleovarans PHA polymerase followed by a selection process that involves detection of accumulation of polymer by phenotypic appearance or increased density have been suggested but not been carried out (Peoples et al., U.S. Pat. No. 5,534,432). A simple and effective method for genetically engineering an extension of substrate specificity in a bacterial PHA polymerases is needed.